Recent vaccination strategies exploit the immunogenicity of viruses or virus-like-particles (VLPs) to enhance the immune response towards antigens. For example, WO02/056905 demonstrates the utility of VLPs as carriers to present antigens linked thereto in highly ordered repetitive antigen arrays. Such antigen arrays can cause a strong immune response, in particular antibody responses, against the antigen, including self antigens. Furthermore, VLPs have been shown to be useful in the therapy of diseases as delivery means for immunostimulatory substances (WO2003/024481). VLPs are therefore useful in the production of pharmaceuticals for the treatment of infectious diseases and allergies as well as for the efficient induction of self-specific immune responses, e.g. for the treatment of cancer, rheumatoid arthritis and various other diseases. For the production of VLP based pharmaceuticals efficient processes for expression and purification of VLPs are required.
For reasons of scalability and efficiency and, thus, cost saving, VLP carriers for the manufacture of pharmaceuticals are preferably produced by recombinant gene expression in a prokaryotic expression system. Viral capsid proteins have been shown to efficiently self-assemble to form VLPs upon expression in a bacterial host. For example, hepatitis B virus derived VLP has been obtained by expression of HBc protein in E. coli and purification of the VLP from bacterial homogenate on a sucrose gradient (WO01/85208). VLPs of bacteriophages, preferably of RNA bacteriophages, are particularly suited as antigen carriers and have been produced in E. coli, wherein the assembled VLPs were isolated from crude bacterial homogenates by various methods.
For example, recombinant VLP derived from bacteriophage fr was isolated from lysed E. coli cells by ammonium sulphate precipitation followed by size exclusion chromatography using a SEPHADEX® G100 column with a SEPHADEX® G25 pre-column (Pushko et al. 1993, Protein Engineering 6(8)883-891). Soluble recombinant VLPs derived from bacteriophage MS-2 were isolated from lysed E. coli cells by a combination of ammonium sulphate precipitation and separation on a sucrose density gradient, while less soluble variants were isolated by size exclusion chromatography (Mastico et al. 1993, Journal of General Virology 74:541-548). WO92/13081 teaches the isolation of MS-2 derived VLP by fractionated ammonium sulphate precipitation combined with either sucrose density gradient separation, gel filtration or immuno affinity chromatography. A multi step purification scheme for recombinant MS-2 derived VLP comprising ammonium sulphate precipitation, isoelectric point precipitation, sucrose density gradient separation and size exclusion chromatography was also reported (Stockley & Mastico 2000, Methods in Enzymology 326:551-569). Recombinant VLPs derived from bacteriophage Qβ have been purified from bacterial homogenate by size exclusion chromatography using a SEPHAROSE® column (Kozlovska et al. 1993, Gene 137:133-137) or by a combination of fractionated ammonium sulphate precipitation and size exclusion chromatography with SEPHAROSE® CL-4B (Vasiljeva et al 1998, FEBS Letters 431:7-11; Ciliens et al. 2000, FEBS Letters 24171:1-4).
Proteins isolated from bacterial homogenates are typically contaminated with endotoxins and other host cell derived impurities, such as host cell DNA and host cell proteins. The presence of host cell derived impurities, especially endotoxins, is generally undesired in pharmaceutical preparations. Endotoxins are lipopolysaccharides which are invariably associated with the outer membrane of gram-negative bacteria, such as E. coli. They show a strong toxic, inflammatory and/or immunogenic effect on mammals, including humans, when entering the blood stream. Thus, removal of even minute amounts of endotoxins from protein preparations used for the manufacture of a pharmaceutical composition is essential. The processes which so far have been applied for the purification of recombinant VLPs from bacterial homogenates are not capable of reliably removing endotoxin contaminants to an extent which is acceptable for pharmaceutical compositions and/or said processes comprise steps, such as sucrose gradient separation, which can hardly be scaled up for commercial production of the VLP.
It is an object of the present invention to provide a process for the purification of recombinantly expressed, self-assembled VLPs from bacterial homogenate, wherein the VLPs are essentially free of host cell derived impurities, especially of endotoxins, and wherein the process can be scaled up to a commercial production scale in a cost effective manner.
The assembly of the VLP takes place in the cytosol of the bacterial host expressing the VLP, while endotoxins are associated with the cell wall of the bacterial host. Therefore, endotoxins are typically not encapsulated inside the VLP and can thus be efficiently removed by the process of the invention. The VLP preparations obtained by the process of the invention typically comprise endotoxin contaminations at concentrations which are about 50 times lower than those observed in preparations obtained by the methods mentioned above.
The invention therefore provides a process for the purification of a VLP from a recombinant bacterial host expressing said VLP, wherein the process is capable of removing endotoxins and that fraction of nucleic acids and host cell proteins which is not encapsulated inside the VLP.